Current pathological indices cannot accurately predict the treatment outcome and the risk of recurrence/metastasis for individual cancer patients. The extent of tumor mitochondrial metabolism and its heterogeneity within the tumor are crucial factors in cancer progression that cannot be adequately determined with existing techniques. There are great interests and unmet needs in testing whether the combination of imaging indices of the tumor mitochondrial metabolism and its heterogeneity can be incorporated into the breast cancer prognostic models to facilitate individualized treatment. The objective of this project is to demonstrate that the mitochondrial redox imaging, i.e., fluorescence imaging of endogenous NADH and oxidized flavoproteins (Fp including FAD), has prognostic value in managing breast cancer (BC). Our central hypothesis is that the heterogeneity-associated mitochondrial redox imaging (HAMRI) indices (Fp, NADH and the redox ratios, e.g., Fp/NADH) can predict or add value for predicting BC prognosis. In the first aim, we will identify the mitochondrial redox imaging indices for BC prognosis in patients. We will develop an optical-imaging needle device and obtain real-time redox imaging measurements in vivo during the biopsy procedures. We will test hypothesis 1a that the HMARI indices can differentiate among cancerous (invasive vs in situ), premalignant and benign/normal breast tissues. We will also conduct redox imaging retrospectively on clinical BC frozen specimens collected since 2008. We will test hypothesis 1b that the HAMRI indices can predict the risk of local recurrence and metastasis of BC and have the potential to add value to current clinical prognostic model. Furthermore, we will test hypothesis 1c that HAMRI can predict nodal status. In the second aim, we will identify the metabolic basis for prognostic redox imaging indices in mouse and cell models. We will test hypothesis 2a that 1) the HAMRI indices correlate with the metastatic potential of BC mouse xenografts in vivo measured by fluorescence imaging of red fluorescence proteins in the metastases, and 2) the HAMRI indices correlate with the invasive potentials of BC cell lines in vitro. We will treat cancer cells with different biochemical reagents to modify th mitochondrial redox state. We will test hypothesis 2b that changes of the mitochondrial redox state correlate with the changes of invasive/metastatic potential of BC. We will also determine if these changes are associated with changes in the levels of free radicals and oxidative damages. Anticipated outcomes from this project include: 1) Establish the HAMRI indices as rapid (real time achievable), low-cost, and independent predictors of BC prognosis; 2) Establish the mitochondrial redox state as a key factor in BC progression to metastasis. These novel results will have significant clinical impact on breast cancer management and can be extended to other diverse forms of cancers. We believe that this research, with further developments, has the potential to reshape the current BC prognostic models and therefore, open new, novel paradigms in the treatment and monitoring of breast cancer.